003 1-399819 1/2906-0601$03.00/0
PEDIATRIC RESEARCH
Copyr~ght(c) 199 1 lntcrnational Pediatric Research Foundation, Inc.
Vol. 29. No. 6. 199 1
I'rin/c~/ in U.S /I
The Incorporation of n-3 and n-6 Essential Fatty
Acids into the Chick Embryo from Egg Yolks
Having Vastly Different Fatty Acid Compositions
DON S. LIN, WILLIAM E. C O N N O R . A N D G R E G O R Y J. A N D E R S O N
i)i~.i.,ror~o ~ ' I : ' r ~ ~ l o c ~ r r lr ~~ior ri o
h c~i~ca~~n.~~d~C, l i ~ i i c ~ h'~~lr.ifiori,
ui
Dcy~u,?mcni oj'Mcdiciric, Orc2~ronIlccilill S c r a ~ t . e s
C n r i.ci:sr/~~.
I~or/lcr~ici,
O r c ~ o n9 72111-3098
ABSTRACT. The effect of egg yolk fatty acid composition
on essential fatty acid utilization by the developing chick
embryo was studied by feeding laying hens a fat-free diet
supplemented with oils containing widely divergent contents of the essential n-6 and n-3 fatty acids. A control hen
was fed a commercial feed for laying hens. The diets
contained 20 to 4370 mg/100 g n-3 fatty acids and 360 to
8020 mg/100 g n-6 fatty acids. Fertile eggs were collected
in pairs: one was incubated and the other served a s an
unincubated control. The fatty acid content of the unincubated egg and the newly hatched chick from each pair was
compared. Some 50% of the total fatty acids in the egg
yolk were incorporated into the tissues of the newly
hatched chick. Regardless of diet, more yolk n-6 fatty acids
were incorporated into the chick compared to saturated or
monounsaturated fatty acids. The percentage of incorporation especially increased from the eggs containing relatively low amounts of n-6 fatty acids. The percentage of
incorporation of n-3 fatty acids was similar to that of
saturated and monounsaturated fatty acids when n-3 fatty
acids were plentiful in the egg yolk, but increased significantly when n-3 fatty acids were low in the eggs. There
was a generally linear relationship between essential fatty
acids in the egg and in the chick, although levels of
docosahexaenoic acid [DHA; 22:6(n-3)] in the brain did not
respond proportionally. The developing chick preferentially removed D H A from the yolk, but did not synthesize
more D H A when the amount of the D H A precursor,
18:3(n-3), in the yolk was increased. W e concluded that
the developing chick embryo requires 0.4-1.1% of egg
energy a s n-3 fatty acids and 4.8-6.2% as n-6 fatty acids,
or a "dietary" ratio of n-6111-3 of 5 to 14. This requirement
may have relevance for humans a s well. (Pediatu Res 29:
601-605,1991)
Abbreviations
DHA. docosahexaenoic acid
The essentiality of n-6 fatty acids was established over 50 years
igo (1, 2). Now a growing body of evidence indicates that n-3
'atty acids have their own distinctive role in the structure and
unction of biologic membranes, particularly in the retina and
I N S (3). Depriving rats of dietary n-3 fatty acids for two generReceived October 30. 19'10: accepted February 13. 1991
Cart-cspondcnce. Willlam E. Connor. M.D.. D c p a ~ t ~ n e noft Medicine. L465.
l i e Oi'cgon Hcalth Sclcnccs University. Portland. O R 07201-3098.
Suppol-tcd by N l l l GI-ants I IL-07295. HL-25687. HL-37940. DK-29930. DK(103.5. and RR-00334 and a rcsearcll fellowship (G J.A.) fioi11 rhe Ainei-ic;in Hcait
saoclatlon. 01-cgonAlliliate.
ations, Yamamoto el a/. (4, 5) and Bourre et a/. (6) reported a
significantly lower learning ability in the deficient animals. Feeding pregnant monkeys a n n-3 fatty acid-deficient diet, we found
that the resulting infants suffered visual impairment, had abnormal electroretinograms, and developed polydipsia (7-10).
Unlike mammals, the chicken has a unique reproductive
system that is self-contained with regard to nutrients. Lipid-rich
egg yolk is the chief source of nutrients for the avian embryo
(1 1). This biologically self-sufficient model allows a close correlation between nutritive substances and their physiologic utilization. In a previous study, we demonstrated that dietary fats of
the laying hen can drastically alter the fatty acid composition of
the egg and, in turn, that of the newborn chick (12). In our
present study, we quantitatively examined the relative contribution of essential fatty acids to embryo formation when the egg
yolk contained vastly differing amounts of both n-6 and n-3 fatty
acids.
MATERIALS A N D M E T H O D S
Five single-comb White Leghorn laying hens (commercial
strain) were housed individually under 16 h light/d as approved
by the University Animal Care Committee. Each hen was fed a
different diet: four of the hens were fed a semipurified fat-free
basal mix (12) (62.53% dextrose, 20.78% fat-free casein, 0.39%
L-arginine, 0.39% DL-methionine, 3.33% cellulose, 12.20% salt
mix, 0.38% vitamin mix, and 10% fat; Teklad, Madison, WI)
containing 10% (wt/wt) of safflower oil, soy oil, linseed oil, or
fish oil (MaxEPA; Seven Sea Limited. Hull, UK), respectively,
and the remaining hen was fed a commercial diet for laying hens
containing 3% fat and consisting principally of corn mash. These
diets differed greatly in their fatty acid composition and content
(Table 1). The safflower oil diet had a very high content of n-6
fatty acids (80% of total fatty acids) and very little of the 11-3
fatty acids (0.2%). At the other extreme, both the fish and linseed
oil diets had a high content of n-3 fatty acids (34 and 44%,
respectively). Fish oil is rich in longer chain ( Z C ~ ~n-3
, ) fatty
acids, but linseed oil contains only linolenic acid [ I 8:3(n-3)]. In
preparing the diets, the various oils were mixed thoroughly with
the powdered basal mixes. Diets were stored at 4°C and made
fresh weekly, and the feed cups were changed daily. Precautions
were taken to minimize oxidation of the polyunsaturated fatty
acids (1 2).
Every other night, a White Leghorn rooster was placed in the
cage with the laying hens for insemination. Fertile eggs were
collected daily from these hens after they had consumed their
respective diets for 1 m o (12), by which time the fatty acid
composition of the eggs had stabilized. Eggs laid on adjacent
days were collected in pairs: one egg was incubated and the other
(unincubated) egg was analyzed as a control to represent the lipid
composition of the egg yolk before incubation. For each diet, six
pairs of eggs were collected. Eggs were incubated in a Marsh
602
LIN ET AL
Roll-X automatic incubator at 98-99°F. The various experimental diets produced no differences in egg production, fertility, or
hatchability.
Chicks were killed within 12 h of hatching and were not fed.
Lipids were extracted from the brain, yolk sac contents, carcass,
and the egg yolk of unincubated control eggs by the method of
Folch et al. (13). The carcass was first cut into small pieces,
homogenized in a Waring blender, and extracted five times.
Antioxidant (butylated hydroxytoluene) was added to all lipid
extracts (14). The fatty acid composition of the various samples
was determined by capillary gas-liquid chromatography as described before (12). For quantitative fatty acid determinations,
heptadecanoic acid (17:O) was added to duplicate aliquots as an
internal standard. Recovery of DHA was verified by parallel
analysis of an external standard. For data reported as whole
chick, the values for the carcass, brain, and yolk sac were added
together.
Percentage of incorporation of a fatty acid into the chick is
defined as the amount of that fatty acid recovered in the newly
hatched chick divided by the amount present in a paired egg that
was not incubated. This calculation does not, however, give a
straightforward measurement of the transfer of fatty acids from
egg to chick. For example, because the chick embryo is able to
desaturate and elongate fatty acids (15, 16), a portion of a
"recovered" fatty acid in the chick might be derived from a
precursor fatty acid in the egg yolk. Similarly, a portion of a
given fatty acid in the egg yolk could be metabolized to longerchain or more unsaturated products. In addition, the developing
chick derives most of its energy from oxidation of yolk fatty
acids ( 1 1, 17). Thus, the "percentage of incorporation" defined
here represents a net balance between anabolic, catabolic, and
transport processes.
Comparison of the effect of diet on the incorporation of fatty
acids from egg to the tissues of the chick was correlated by
analysis of variance. Comparison of the incorporation of saturated, monounsaturated, n-6, and n-3 fatty acids within a given
diet was performed by repeated measures analysis of variance.
Differences between individual means were detected by use of
the appropriate t statistic (18), using the Bonferroni inequality
( 1 9) to control the overall a-level.
RESULTS
The relative conservation of essential fatty acids during the
utilization of egg yolk lipids by the developing chick embryo can
be seen in Table 2. Regardless of the essential fatty acid content
of the diet, a higher percentage of total n-6 fatty acids, as opposed
to either saturated or monounsaturated fatty acids, was incorporated into the chick ( p < 0.02). The n-3 fatty acids were also
preferentially incorporated, but only in the two diet groups
(control and safflower oil) that had the lowest levels of n-3 fatty
acids in the egg. In general, changing the amount of total n-6
and total n-3 fatty acids in the egg led to reciprocal effects on the
incorporation of n-6 and n-3 fatty acids. Thus, when n-6 fatty
acids in the egg were low and n-3 fatty acids were high (fish and
linseed oil diets), incorporation of total n-6 fatty acids was high
(64%) and incorporation of total n-3 fatty acids was lower (54
and 51%, respectively). And when n-3 fatty acids were low
(control and samower oil diets) incorporation of n-3 fatty acids
was high (66 and 74%, respectively). This reciprocal effect was
particularly pronounced in the case of 20:4(n-6) and DHA, where
the feeding of the fish and safflower oil diets caused so little
20:4(n-6) and DHA, respectively, to be deposited in the egg that
the developing chick retained >80% of the fatty acids originally
present in the yolk. Of course, some of this apparent incorporation of these long-chain polyunsaturates could have been due to
synthesis from 18 carbon precursors, i.e. 18:2(n-6) and 18:3(n3). This is likely not the sole explanation, inasmuch as the
amount of 18:2 and 18:3 incorporated into the fish oil and
samower oil chicks, respectively, was not low relative to the other
diets. An effect of diet on the apparent incorporation of 22:4(n6) clearly was due to synthesis from precursors, however. In this
case, more 22:4(n-6) was recovered in the chick than was present
in the egg, resulting in an incorporation rate greater than 100%.
The incorporation of total n-6 and total n-3 fatty acids into
the chick is displayed graphically in Figure 1, where the trend
toward a reciprocal relationship between diet and incorporation
can be seen clearly.
Despite differences in the percentage of incorporation of essential fatty acids into the chick, there was a generally linear relationship between the amount of n-6 and n-3 fatty acids in the
egg and in the chick (r = 0.83, p < 0.001 for n-3; r = 0.93, p <
0.001 for n-6). However, not all chick tissues responded to higher
amounts of n-3 fatty acids in the egg. Brain levels of DHA did
not respond proportionally when the amount of DHA in
the egg was increased beyond that found in the eggs produced
on the soybean oil diet. This is shown graphically in Figure 2,
where the amount of DHA in the egg is displayed in conjunction
with the amount of DHA found in the whole chick and in the
brain alone.
The extremely high levels of 18:3 in the eggs of hens fed the
linseed oil diet did not lead to any real increase in the amount
of DHA found in the chick compared to eggs from the soy diet
(Fig. 3A). In fact, the very small increase in whole chick DHA
that was measured in the linseed versus the soy chicks could be
attributed to the slightly higher levels of "preformed" DHA in
the eggs of the linseed oil hens (Fig. 3B).
The utilization of fatty acids by the developing chick embryo
was further studied by examining the residual yolk contained in
the yolk sac of the newly hatched chick. The fatty acid composition of this material is compared in Table 3 with that of the
control unincubated egg yolk. It is apparent that DHA and 20:4
were preferentially absorbed from the yolk during incubation.
This effect was seen in all five diets.
Table I . h t t y ucid content jmg/l00 g diet) andfatty acid composition (weight %j of experimental diets*
Total fatty acid
Saturated
Monounsaturated
Polyunsaturated
Total n-6
18:2 (n-6)
20:4 (n-6)
Total n-3
18:3 (n-3)
20:5 (n-3)
22:5 (n-3)
22:6 (n-3)
Ratio n-6/n-3
Control
Safflower oil
Soy oil
Fish oil
Linseed oil
2976
405 (13.5)
759 (25.3)
18 12 (60.4)
1764 (58.8)
1746 (58.2)
10010
890 (8.9)
I080 (10.3)
8040 (80.4)
8020 (80.2)
7990 (79.9)
9980
1610 (16.1)
2280 (22.8)
61 10 (61.1)
5450 (54.5)
5450 (54.5)
9940
1170 (1 1.7)
2230 (22.3)
6570 (65.7)
2170 (21.7)
2170 (21.7)
48 (1.6)
42 (1.4)
20 (0.2)
20 (0.2)
630 (6.3)
630 (6.3)
36.8
40 1
9590
2970 (29.7)
2840 (28.4)
3780 (37.8)
360 (3.6)
170(1.7)
I10 (1.1)
3420 (34.2)
70 (0.7)
1640 (16.4)
280 (2.8)
1 I00 (1 1.0)
0.1
* Fatty acid composition (%) is given in parentheses
8.7
4370 (43.7)
4370 (43.7)
0.5
604
LIN ET AL.
Linseed
Soy
Fig. 3. Effect of egg 1 8 3 ( A ) and DHA (B) levels on the amount of
DHA deposited in the chick (means + SD).
DHA, for growth and membrane maintenance. When a fat-free
or fat-deficient diet is eaten, the body attempts to compensate
for the lack of n-6 and n-3 polyunsaturated fatty acids by
synthesizing a polyunsaturated fatty acid of the n-9 family,
namely 5,8,11-eicosatrienoic acid [20:3(n-9)]. When animals are
fed a diet deficient in n-3 fatty acids, the body compensates for
the lack of needed DHA by increasing the synthesis of a longchain polyunsaturated fatty acid of the n-6 family, namely
4,7,10,13,16-docosapentaenoic
acid [22:5(n-6)].In our study, the
developing chick embryo responded to a short supply of essential
n-6 or n-3 fatty acids in the yolk by increasing the percentage of
the scarce fatty acid that was incorporated from the egg. Thus,
less of the fatty acid in short supply was shunted to oxidative
pathways to supply energy for embryo formation and more was
incorporated into the tissues. It has been estimated that in excess
of 90% of the energy requirement of the chick embryo is derived
from oxidation of yolk lipid fatty acids during development,
there being little carbohydrate in eggs (1 1, 17).
This diet-induced difference in the metabolism of fatty acids
by the embryo is in addition to the diet-independent preference
for sparing of certain fatty acids, especially n-6 fatty acids. Leyton
et al. (20) have also found a difference in the treatment of various
fatty acids by the whole body. In their study, rats oxidized fatty
acids differently, depending generally on chain length and degree
of unsaturation. This differential oxidation phenomenon may be
a contributing factor to net incorporation observed from egg to
chick in the present study.
Comparing the fatty acid composition of egg yolk triglyceride
and phospholipids with that of the 20-d chick embryo, Isaacks
et a/. (21) concluded that the polyunsaturated acids of egg yolk
triglyceride and phospholipid appear to be preferentially utilized
in the development of the chick embryo. Analyzing the fatty
acid composition of various phospholipids in the unincubated
egg and in eggs incubated for 13, 15, 17, 19, and 21 d, Noble
and Moore (22) suggested that the developing embryo preferentially absorbs from the yolk a phosphatidylethanolamine fraction
that is relatively rich in DHA. On the other hand, Bordoni et al.
(23) concluded that lipid absorption did not favor any fatty acid.
In our study, we found preferential absorption of both arachidonic acid and DHA from yolk lipids during chick embryo
formation under a variety of dietary conditions.
The content of essential n-6 and n-3 fatty acids in the egg and,
in turn, in the chick was proportional to the amounts in the diet
of the laying hen. However, although the essential fatty acid
content of the diet of the laying hen had a significant effect on
the fatty acid composition of the brain of the newborn chick,
this effect was not proportional. For example, greatly increasing
the amount of DHA or DHA precursor in the egg led to only a
moderate increase in brain DHA. Undoubtedly, the phospholipid
membranes of the normal brain have an appropriate fatty acid
composition, and a large increase in the proportion of DHA may
not be compatible with optimal functioning. This optimal fatty
acid composition is probably regulated through selective metabolism of given fatty acids or regulation of uptake into tissues
such as the brain (24).
Despite the great increase in levels of 18:3(n-3) in the tissues
of the linseed oil chicks, no meaningful increase was seen in
tissue levels of the 18:3(n-3) metabolite DHA. The small increase
in tissue DHA that was observed in the linseed oil chicks could
be completely explained by the small increase in preformed DHA
that was transferred into the egg yolk by the hens fed the linseed
oil diet. This is consistent with studies in the growing chick (25)
that showed dietary 18:3(n-3) to be less effective than preformed
DHA at supporting deposition of DHA in nervous tissue.
The dietary requirement for n-3 fatty acids has been a matter
Table 3. Effect of diet on fatty acid cornposltion (welght %) of egg yolk and chick yolk sac contents*
Control
(n = 4)
Yolksac
Egg
Egg
Yolksac
Yolksac
35.9 i 2.5 35.2 + 1.5 34.4 + 1.8 35.5 k 0.8 35.7 + 0.8
47.7 + 1.2 28.5 + 1.3 29.6 + 1.0 3 6 . 9 k 0.6 38.5 +- 0.5
16.2 11.5
15.4 +- 1.3 36.0 i 0.9
15.2 t 1.5
12.8 +- 0.5
1.8iO.l
0.1t0.0
0.3 + 0.2
0.9k0.1
0.3r0.0
0.1 + 0.1
0.5k0.1
Egg
14.4 + 1.1
14.4 1.3 35.8 0.9 34.8 2.9 24.4 -t 0.7
12.8 k 0.6 31.9 + 0.6 32.2 + 1.9 22.0 k 0.8
1.2k0.2 2.550.2
1.6k0.2 2.1ko.l
O.lk0.0 0.2+0.0
0.1+0.1
O.lt0.l
0.4 + 0. l
0.1 + 0.1
0.1 t 0.0
0.620.2
0.2+0.1
O.l+O.l
2.6zkO.l
0.3+0.1
0.1+0.0
0.10.0
1.3kO.l
24.0 +- 0.8
22.0+- 0.8
1.4+-0.1
O.lk0.1
1.220.1
0.1iO.1
0.2i0.1
2.9 + 0.6
1.9 + 0.6
0.7k0.0
0.2+-0.0
0.1 k 0 . 1
11.5f0.7
0.3k0.l
2.5t0.2
1.8k0.2
6.7+0.6
0.2+-0.1
+
0.1+0.0
+
0.0+0.0
* Means +- SD.
t Yolk sac different from egg yolk for all diets, 11 < 0.001
1.7f0.1
1.4i0.1
Yolksac
Linseed oil
(n = 6)
Egg
Yolksac
43.1 -t 4.2 40.8 + 1.6 36.5 zk 1.2 36.1 -c 1.8
43.8 + 0.4 47.2 + 1.2 39.3 -C 1.1 41.2 + 1.0
2 7 . 0 k 0.8 25.9 + 1.2
+
35.2 + 2.3
Fish oil
(n = 6)
Soy oil
(n = 5)
38.8 k 0.9
45.5 + 0.6
Egg
Saturated
Monounsaturated
Polyunsaturated
Total n-6
18:2 (n-6)
20:4 (n-6)t
22:4 (n-6)
22:s (n-6)
Total n-3
18:3 (n-3)
20:s (n-3)
2 2 5 (n-3)
22:6(n-3)l.
Safflower oil
(n = 6)
1 1 . 1 k 0.8 22.8
2.9 k 0.7
2.1 +- 0.5
0.6tO.l
0.2+-0.1
7.7+0.7
0.3+0.l
2.1k0.1
1.7+0.2
3.4-tO.6
* 0.9
10.5 k 0.4
9 . 3 k 0.1
0.8+0.l
0.lko.l
0.5 + 0.1
12.0k0.4
9.1k0.4
0.4k0.1
0.5kO.1
1.9zk0.3
+ 1.6
10.6 + 0.7
9.5 + 0.6
21.3
0.6+-0.1
0.1-tO.l
10.8+1.0
9.4k1.0
0.2i0.l
0.4+0.1
0.6k0.1
YOLK FATTY ACIDS AND THE CHICK EMBRYO
of some discussion, with little hard data (26,27). Our observation
of a relative sparing of n-3 fatty acids during development of
chicks from the low n-3 fatty acid groups (control and safflower
oil diets) may mean that n-3 fatty acid levels in these two diets
were below the "requirement" for the developing chick. Thus,
these chicks may have metabolically sensed that the essential n3 fatty acids were in short supply, and may have attempted to
conserve scarce resources by channeling less of these fatty acids
into oxidative pathways. The lack of a sparing response in chicks
from hens fed the soybean oil diet may mean that the amount
of n-3 fatty acids in the egg yolk met or exceeded the requirement.
Thus, one can estimate the requirement for n-3 fatty acids in the
developing chick embryo as being between the levels of n-3 fatty
acids present in eggs containing: I ) the lowest amount of n-3
fatty acids that did not evoke a sparing response and 2) the
highest amount of n-3 fatty acids that did evoke a sparing
response. This would correspond to the soybean and control
diets, containing an average of 84 and 26 mg of n-3 fatty acids
per egg, respectively. With a calculated average of 65.9 kcal per
egg in our study (28), and assuming 9 kcal/g fatty acid, the
amount of n-3 fatty acids in the soybean and control diets equates
to 1.1 and 0.4% of energy, respectively. Thus, the requirement
for n-3 fatty acids can be estimated as being between 0.4 and
1.1% of energy.
For n-6 fatty acids, a sparing response was observed in chicks
fed the fish and linseed oil diets. By the same reasoning as given
above for n-3 fatty acids, the estimated requirement for n-6 fatty
acids lay between the linseed and control diets, equating to 4.8
to 6.2% of energy, respectively. This is somewhat higher than
3.8% of energy, the requirement for linoleic acid measured in 1to 3-wk-old chicks from hens fed diets low in linoleic acid (29).
These values for n-3 and n-6 requirements for the developing
chick would suggest an optimal "dietary" ratio of n-6 to n-3 fatty
acids of between 5 and 14.
This study may have several possible implications for the diet
of human infants. The first is that the ratio of n-6 to n-3 fatty
acids is a useful way of expressing a dietary recommendation
because this ratio appears to have a relationship to DHA concentrations in the brain from the data in this paper. Very high ratios
(>150:1), as in diets based on safflower, sunflower, or peanut
oils, produce DHA tissue depletion and retinal abnormalities in
animals (3), and high ratios in the diets of human infants may
reduce visual acuity and lead to abnormalities in the electroretinogram (30). Secondly, the absolute amounts of n-3 fatty acids
in infant formulas are important. Our view is that the n-3 fatty
acids should approximate the amounts and kinds present in
human milk. In human milk, n-3 fatty acids (including small
amounts of preformed DHA) normally represent 0.7-1.3% of
calories, and the n-6 to n-3 ratio is 4:l to 10:l (31, 32). We
would suggest that these values be used as a guide for the
preparation of human infant formulas, and that DHA should be
included. Retinal and brain DHA accumulation may not be
optimal when only a-linolenic acid [18:3(n-3)J is present in the
diet. It should be noted that several currently marketed infant
formulas fail to meet the n-6111-3 ratio criteria and none of them
contain any DHA. We appreciate the controversial aspects of
these suggestions and anticipate that there will not be universal
agreement. There was, however, a general consensus of views
similar to ours at a recent meeting about n-3 fatty acid requirements (33).
Acknowledgments. The authors thank Julie D. Corliss for
technical assistance and David Wilson and Dr. Gary J. Sexton
for help with statistical analysis of the data.
REFERENCES
I. Burr GO. Burr MM 1929 A new deficiency disease produced by the rigid
exclusion of fat from the diet. J Biol Chem 82:345-367
605
2. Burr GO, Burr MM 1930 On the nature and role of the fatty acids essential in
nutrition. J Biol Chem 86:587-621
3. Neuringer M. Anderson GJ, Connor WE 1988 The essential~tyof n-3 fatty
acids for the development and function of the retlna and brain. Annu Rcv
Nutr 8:5 17-54 1
4. Yamamoto N. Saitoh M, Morlilchi A. Nomura M. Okuyama H I987 Eff'ect
of dietary n-linolcnatc/l~noleatebalance on brain lipid composit~onsand
learning ability of rats. J Lipid Res 28: 144-1 5 1
5. Yamamoto N, Hashimoto A, Takemoto Y. Okuyama 1-1. Nomura M. Kltayima
R. Togashi T. Tamai Y 1988 Effect of the dietary e-l~nolenate/linoleate
balance on lipid compositions and learning ability of rats. 11. Discrimination
process, extinction process. and glycolipid compositions. J Lipld Res
29:1013-1021
6. Bourre JM. Francois M. Youyou A. Dumont 0. Piciotti M. Pascal G. Durand
G 1989 The effects of dietary alpha-Ilnolenic acid on thc composition of
ncrve membranes. cnzymatlc activity, amplitude of electrophys~ological
parameters. resistance to poisons and performance of learnlng tasks in rats.
J Nutr 119:1880-1892
7. Neuringer M, Connor WE, Lin DS. Barstad L. Luck SJ 1986 Biochemical and
functional effects of prenatal and postnatal omega-3 fatty acid defic~cncyon
retina and brain in rhesus monkeys. Proc Natl Acad Sci USA 83:4021-4025
8. Neuringer M, Connor WE, Van Patten C, Barstad L 1984 Dietary omega-3
fatty acid deficiency and visual loss in infant rhesus monkeys. J Clin Invest
77:272-276
- - - - .
9. Connor WE, Neuringer M. Barstad L. Lin DS 1984 Dietary deprivation of
linolenic acid in rhesus monkeys: effects on plasma and tissue fatty acld
composition and on visual function. Trans Assoc Am Physicians 97: 1-9
10. Reisbick S. Neuringer M, Hasnain R, Connor WE I990 Polydipsia In rhesus
monkeys deficient in omega-3 fatty acids. Physiol Behav 47:3 15-323
I I. Romanoff AL I967 Blochemistry ofthe avian embryo. John Wiley. New York
12. Anderson GJ. Connor WE. Corliss JD, Lin DS 1989 Rapid modulation of thc
n-3 docosahexaenoic acid levels in the brain and retina of thc newly hatched
chick. J Lipid Res 30:433-441
13. Folch J, Lees MB, Sloane-Stanley G H 1957 A simple method for the isolation
and purification of total l~pidsfrom animal tissues. J Blol Chem 226:497509
14. Miljanich GP, Sklar LA, White DL, Dratz EA 1979 Disaturated and d~polyunsaturated phospholipids in bovine retinal rod outer segment d ~ s kmembrane.
Biochim Biophys Acta 552294-303
15. Miyamoto K, Stephanides LM, Bernsohn J 1967 Incorporation of [I-14C]
linoleate and linolenate into polyunsaturated fatty acids of phospholipids of
the embryonic chick brain. J Neurochem 14227-237
16. Noble RC, Shand JH 1985 Unsaturated fatty acid compositional changes and
desaturation during the embryonic development of the chicken (gallus
domesticus). Lipids 20:278-282
17. Freeman BM, Vince MA I974 Development of the avian embryo. Chapman
and Hall, London
18. Wlner BJ 1971 Statistical Principles In Experimental Deslgn. McGraw-Hill.
New York
19. Miller RG 1966 Simultaneous Statistical Inference. McGraw-Hill. New York
20. Leyton J, Drury PJ, Crawford MA 1987 Differential oxidation of saturated
and unsaturated fatty acids rn vivo In the rat [published erratum appears in
Br J Nutr 1987 Sep:58(2):following 3311. Br J Nutr 57:383-393
21. lsaacks RE, Davies RE, Ferguson TM, Reiser R. Couch JR 1964 Studies on
avian fat composition. 2. The selective utilization of fatty acids by the chick
embryo. Poult Scl 43: 1 13- 120
22. Noble RC, Moore JH 1965 Metabolism of the yolk phospholipids by the
developing chlck embryo. Can J Biochem 43:1677-I686
23. Bordoni A, Cocchi M , Lodi R, Turchetto E, Ruggeri F 1986 Metabolism and
distribution of linoleic and LY-linolenicacid derivatives in chick cmbryo
development. Prog Lipid Res 25:407-41 I
24. Anderson GJ. Connor WE 1988 Uptake of fatty aclds by the developing rat
brain. Lipids 23:286-290
25. Anderson GJ, Connor WE, Corliss J D I990 Docosahexaenoic acid is the
preferred dietary n-3 fatty acld for the development of the brain and retina.
Pediatr Res 27:89-97
26. Simopoulos AP I989 Summary of the NATO advanced workshop on dietary
omega 3 and omega 6 fatty acids: biological effects and nutritional essentiality. J Nutr 119:521-528
27. Food and Nutrition Board (National Research Council) 1989 Recommendcd
Dietary Allowances. Natlonal Acadcmy Press, Washington, DC
28. Pennington JAT I989 Bowes & Churche's Food Values of Portions Comn~only
Used. Lippincott, Philadelphia
29. Hopkins DT, Nesheim MC 1967 Thc linoleic acid requirement ofchicks. Poult
Sci 46:872-881
30. Uauy RD. Birch DG, Birch EE. Tyson JE, Hoffman DR 1990 Effect of dietary
omega-3 fatty acids on retinal function of very-low-birth weight nconatcs.
Pediatr Res 28:485-492
3 1. Gibson RA. Kneebone G M 198 1 Fatty acid composition of human colostrum
and mature breast milk. Am J Clin Nutr 34252-257
32. Jansson L. Akesson B, Holmberg L 1981 Vitamin E and fatty acid composition
of human milk. Atn J Clin Nutr 34:8-13
33. Simopoulos AP (cd) 1991 Health effects of omega-3 polyunsaturated fatty
acids in seafood. World Rev Nutr Diet 66: 1 1 8-132